Recombinant therapeutic proteins are increasingly important to the pharmaceutical industry. They represent valuable treatments against life threatening disease as well as investment opportunity for many biopharmaceuticals. Total global spending on medicines is estimated to reach $1 trillion by 2017, surpassing the 2012 spending level of $205-$235 billion. Surprisingly, data suggests that this growing share among all medicines class are biologic, with biosimilars or non-original biologics, making up about 20% of global pharmaceuticals market. These recombinant therapeutic proteins are divided into many sub classes such as hormones, monoclonal antibodies, fusion proteins, antibody fragments, vaccines, etc. . . . In general, these class of therapeutic proteins are expressed in a mammalian cells in a medium which provides all nutritional requirement to help cell growth and expression of these recombinant proteins. In the past, many recombinant therapeutic proteins approved by the FDA are expressed in variety of cells such as, but are not limited to, Chinese Hamster Ovary (CHO), Human Embryonic Kidney 293 cells, NS0, etc. . . . CHO cells have been repeatedly approved by regulatory agencies to manufacture these recombinant proteins. CHO cells' ability to culture easily in suspension and to produce high titers of human-compatible therapeutic proteins make CHO cells a popular choice as a host cells to produce these proteins at large scale.
CHO cells were first isolated in 1957 and its genomic resources are presently available. Parental CHO cell line was not able to produce many recombinant proteins at high titer and thus, many CHO sub lineage cell lines such as CHO-K1, CHO-DUX B11, CHO-S and DG44 were developed to achieve high titer and protein quality by random cell-line mutagenesis, media optimization and clonal selection. CHO-K1, which was derived from parental CHO cell line, contains a slightly lower amount of DNA than the parental CHO. CHO-K1 was mutagenized to generate CHO-DUX-B11 (also referred to as CHO-DUKX), a cell line lacking DHFR activity. These cells have a deletion of one DHFR allele and a missense mutation in the other. Subsequently, the Proline-dependent CHO-pro3-strain, another derivative of the original CHO cell line, was mutagenized to yield CHO-DG44, a cell line with deletions of both DHFR alleles.
It has also been reported that extensive mutagenesis and clonal selection of original CHO cells result in many missing genes in different CHO cell lines lineages, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this genetic diversity highlight that these cell lines are having different nutritional and metabolic requirements, different growth characteristics patterns and different ability to produce correctly folded and glycosylated protein in-vitro. Therefore, growth of these cells, expression and quality of recombinant proteins expressed in these cells depend on the culture medium composition and process conditions in which cells are expanded and maintained during the production phase of these proteins. This has been a challenge to scientists as they need either to develop the specific medium for their product expressed in these CHO sub lineage cells or screen out the wide range of commercially available mediums which is a time consuming and labor intensive task. Nutritional and metabolic requirements of cells in a transfected pool cells during clone development may also be different due to functionally heterogeneous or clonal variations. Apart from the different nutritional requirement for cell growth, the quality of expressed protein also depends on culture medium composition and conditions. Many therapeutic biologics products require glycosylation for their functionality which helps to increase the pharmacokinetics property and the half-life of recombinant therapeutic protein in the blood. Glycoproteins expressed in these CHO cells exhibits variation in glycan profile and sialylation. In general, glycosylation is an enzymatic site directed, post translational process occurs mainly in Endoplasmic reticulum organelle of a cell. In particular, a glycan composition sugar is transferred enzymatically and attached via a glycosidic bond in both branch or unbranch manner to the proteins, lipids or other molecules. pH stability of culture medium at large bioreactor scale may affect the glycosylation level of expressed recombinant protein in CHO cells. Thus, a successful mammalian manufacturing cell culture process depends on sufficient expression, glycosylation and correctly folded recombinant product which in turn largely depends on culture medium composition and its process conditions. The development of cell culture media formulations and their compositions have been well documented in literature, and a number of media are commercially available. Consequently, a great variety of different cell culture media have been developed. In early cell culture work, developed general chemical mediums largely depends on serum to provide cells nutritional requirements. Since serum has a significant number of disadvantages in manufacturing of bio therapeutics recombinant proteins such as undefined compositions, being the source of many pathogen, variability between batches, labor intensive purification process and its high cost. Therefore, serum became an unfavorable choice to scientists. A number of culture media offer serum replacement formulation. These media also suggest adding animal or plant extracts. However, the use of animal protein supplements in cell culture media also has certain drawbacks. For example, there is a risk that the culture medium and/or products purified from it may be immunogenic, particularly if the supplements are derived from an animal different from the source of the cells to be cultured. If biological substances intended to be used as therapeutics are purified from such culture media, certain amounts of these immunogenic proteins or peptides may be co-purified and may induce an immunological reaction, up to and including anaphylaxis, in an animal receiving such therapeutics.
Presently, there is a focus on the development of chemically defined media which ideally are deprived of serum, serum proteins, plant or animal proteins, plant or animal tissue/organ extract, lipids, steroids, antibiotic and soy hydrolysates. Different types of medium have been attempted such as serum free, protein free or chemical defined medium with a general focus to supplement the nutrients to a wide variety of cells for cells growth and expression of recombinant proteins. These media may be optimized to obtain a desired recombinant clone (CHO cell's sub lineage cell lines) by either supplementing with specific components or developing a feeding compositions. Further, many proteins such as insulin, transferrin, growth factors or animal/plant hydrolysates or other components may be added to optimize the cell growth of interest of clone. These suggested formulations seek to improve the cell growth and productivity in general. Specific aspects such as particular trace ions, carbohydrates or the content of specific amino acids in combination with additional features, replacement of insulin or transferrin, phospholipid precursors, and Na+/K+ ion channel have also been suggested.
Thus, it is clear that due to diversity of cell lines, production processes in use, the large number of media components involved, and the fact that many of those components are interdependent on each others because of the complexity of cellular metabolic pathways, media requirements for two different processes may differ greatly even when all other aspects of those processes are very similar. Even cell lines derived from a common parent often exhibit different nutritional requirements. For medium manufacturers, the requirements become even more diverse by the number of different cell line varieties and processes in common use today. For example, we wouldn't expect a medium designed for the batch culture of CHO cells to be the best medium for a fed-batch culture of mouse myeloma (NS0) cells. The nutritional requirements of those different cell lines and sub lineage cell lines are dissimilar, as are the requirements of different processes (batch and fed-batch).
Accordingly, there remains a need for a chemically defined, serum-free, protein-free, lipid-free, steroid-free, animal component-free and plant component-free, universal production medium which facilitates the growth of all sub lineage cell lines of CHO cells to high density, increases the level of glycosylation and production of correctly folded recombinant protein without the need of further medium optimization and adaptation.